human cd4 t cells  (ATCC)

Journal: Journal of Human Immunity

Article Title: Refractory infantile IPEX with Treg-restricted FOXP3null expression caused by a novel variant in FOXP3

doi: 10.70962/jhi.20250249

Figure Lengend Snippet: Patient CD4 + Tregs demonstrate no meaningful expression of FOXP3. (A) Flow cytometry analysis of patient PBMC and healthy control show CD4 + and CD8 + T cells within the CD3 + compartment (first row), Treg phenotype (CD25 + CD127 − ) (second row), expression of FOXP3 (third row), Helios (fourth row), and TIGIT (fifth row) within the Tregs. (B) FOXP3 expression in the CD4 + Teffs and Tregs, represented as histograms. (C) Quantification of TSDR-demethylated cells in CD3 + T cells (left) and CD4 + T cells (right) from patient samples and male healthy controls (adults, n = 33 and pediatric controls, n = 11) as well as the patient’s newborn screening dried blood spots (DBSs). (D) Kinetics of the percentage of TSDR/CD4 demethylated cells in patient blood since birth. ( E ) Detection of TSDR-demethylated cells within different CD4 + T effector compartments. GD-253 (IPEX patient [Pt]), GD-260 (patient’s mother), and HD-2417 and HD-2645 (healthy donors/controls). Gating strategies for are in .

Article Snippet: CD4 + T cells were isolated from freshly isolated PBMC using human CD4 + T cells Isolation kit (130-096-533; Miltenyi) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Control

Journal: Journal of Human Immunity

Article Title: Refractory infantile IPEX with Treg-restricted FOXP3null expression caused by a novel variant in FOXP3

doi: 10.70962/jhi.20250249

Figure Lengend Snippet: Activated patient CD4 + T cells express FOXP3. (A) Expression of FOXP3 in baseline and activated CD4 + T cells (first row) showing total FOXP3 (clone: 259D) and CTLA4 (second row) as well as total FOXP3 (clone: 259D) and full-length FOXP3 (clone: 150D) (third row) expression by flow cytometry in patient’s mother, healthy control, and patient (Pt) CD4 + T cells. (B) Western blot analysis of FOXP3 expression in the activated CD4 + T cells, showing the full-length and delta2 isoforms of the FOXP3 protein along with the quantification of the protein levels. Source data are available for this figure: .

Article Snippet: CD4 + T cells were isolated from freshly isolated PBMC using human CD4 + T cells Isolation kit (130-096-533; Miltenyi) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Control, Western Blot

Journal: medRxiv

Article Title: Vδ2 T cell activation by malaria is enhanced in second infection via the cell extrinsic cytokine milieu

doi: 10.64898/2026.04.29.26352021

Figure Lengend Snippet: A ) Vδ2 and Vδ1 T cell expression of HLA-DR and CD86 during CHMI in PBMCs at infection (I, 0 days post infection (d.p.i.)), treatment (T, 8 d.p.i.), 7 (T+7, 15 d.p.i.) and 21 (T+21, 29 d.p.i.) days post-treatment ( n = 12). ( B ) HLA-DR, CD86 and CD40 expression on Vδ2 T cells from malaria-naïve donors ( n = 15) ex vivo and after culture for 5 days with media alone, HMBPP, uRBC lysate and pRBC lysate. ( C ) Activation of isolated naïve CD4 T cells with γδ T cells ( n = 6) isolated ex vivo , or after 3-day culture with media, uRBC lysate or pRBC lysate. Positive control is CD4 T cells with CD3/CD28 beads and IL-12, IL-23 and TGF-β (B+C). Frequency of divided cellS in CD4 T cell population after co-culture is shown. ( D ) Phagocytosis of uRBC or pRBC stained with Cell Trace Violet in Vδ2 T cells (n=6) from PBMCs at T+7 during CHMI. Centre lines represent median, box limits indicate the upper and lower quartiles, whiskers extend to 1.5 times the interquartile range. Lines represent paired observations. P from paired data are Wilcoxon signed rank tests. Unpaired comparisons made with Mann-Whitney U test. See also Supplementary Figures 6-9.

Article Snippet: For the mixed lymphocyte reaction assay of γδ and naive CD4 T cells, cells were isolated from PBMCs using the TCRγ/δ+ T Cell Isolation Kit (Miltenyi Biotec; Cat. #130-092-892), Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec; Cat. #130- 094-131), a QuadroMACST M Separator (Miltenyi Biotec; Cat. #130-090-976), pre-Separation Filters (Miltenyi Biotec; Cat. #130-041-407) and LS Colums (Miltenyi Biotec; Cat. #130-042- 401) as per manufacturer’s instructions. γδ T cells were isolated from malaria-naïve PBMCs (ARC) co-cultured with blank media, 10 6 uRBCs or 10 6 pRBCs for 3 days (ensure high effector cell viability) at 37°C, 5% CO 2 in R10.

Techniques: Expressing, Infection, Ex Vivo, Activation Assay, Isolation, Positive Control, Co-Culture Assay, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: (a) PBMC from healthy donors were cultured with BSA-Alexa 647 in the presence of supernatants from 293T cells transiently transfected with HIV-1 molecular clone NL4-3 (clade B, CXCR4 tropic) for 2 h at 37°C. BSA uptake into gated CD4+ and CD8+ T cell populations was determined by flow cytometry. Representative flow cytometry histograms are shown at left. The graph at right shows the fold increase in probe uptake in HIV-1-treated versus untreated T cells, based on median fluorescence intensity (MFI), from independent donors. Mean ± 1 SEM fold increases in probe uptake are depicted. Statistical significance was determined using a Mann-Whitney U test. (b) Microscopic analysis of resting PB T cells cultured or not with NL4-3 virus-containing supernatants in the presence of BSA-Alexa 647 for 2 h. Cells were counterstained with anti-CD4 or anti-CD8 antibodies to distinguish CD4+ and CD8+ T cells. Left, Maximal Intensity Projection (MIP) images are shown. Asterisks indicate CD4+ T cells. Scale bar = 10 µm. Right, violin plot showing the number of macropinosomes per cell (n=115, 368, 81, and 50 for CD4+ untreated and NL4-3 treated, and CD8+ untreated and NL4-3 treated, respectively). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction. (c) Purified resting CD4+ T cells cultured or not with NL4-3 virus-containing supernatants in the presence of BSA-Alexa 647 for 2h were counterstained with an antibody against HIV-1 p24 capsid protein. MIP images are shown at left. Images on the right show select single slices of the NL4-3-treated samples to identify points of colocalization of BSA and p24. Graphs below show the intensity of green BSA and red p24 signals within the indicated windows in respective slices. Scale bar = 10 µm. (d) Fold induction of BSA uptake in resting CD4+ T cells within PBMC incubated with NL4-3 virus-containing 293T supernatants (crude), NL4-3 virus purified by ultracentrifugation through sucrose (pellet), virus-depleted supernatants (supernatant), or pellet plus supernatant. Probe uptake was determined by flow cytometry. Results from independent donors are depicted. Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction, comparing each sample type to the crude virus as a reference. (e) The flow cytometry histogram shows BSA uptake by resting CD4+ T cells within PBMC in the presence of supernatants from 293T cells transfected with wild-type NL4-3 or NL4-3 lacking the Env glycoprotein. The graph shows the percent of probe uptake by NL4-3 lacking Env compared to wild-type NL4-3 from independent donors. Statistical significance was determined using a Student’s 1-sample t-test.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Cell Culture, Transfection, Flow Cytometry, Fluorescence, MANN-WHITNEY, Virus, Purification, Incubation

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: PBMC (a, c-e) or purified CD4+ T cells (b) were incubated with BSA-Alexa 647 and soluble recombinant gp120 from HIV-1 strains IIIB (clade B, CXCR4 tropic) or BAL (clade B, CCR5 tropic) at 2 µg/ml (a,b,d,e) or at the concentrations indicated (c) for 2 h at 37°C (a-c), or for the time indicated at 37°C (d), or for 2 h at the temperatures indicated (e). Flow cytometry histograms show representative probe uptake in CD4+ and CD8+ T cells (a). Graphs show fold increases in BSA uptake in CD4+ and CD8+ T cells from independent donors. Statistical significance was determined using a Mann-Whitney U test (a,c,d) or Student’s 1-sample t-test (b,e). (f) Microscopic analysis of resting CD4+ T cells following treatment with soluble HIV-1 Env in the presence of BSA-Alexa 647. Representative images are shown on the left; quantitation is shown on the right (n= 27, 129, 212 for untreated, IIIB, and BAL, respectively). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction. (g) Resting and PHA-activated PBMC were incubated with gp120 IIIB or BAL (2 µg/ml) in the presence of BSA-Alexa 647 for 2 h at 37°C. Representative histograms showing probe uptake in CD4+ and CD8+ T cells are shown at top. Graphs depicting fold increase in BSA uptake relative to unactivated CD4+ and CD8+ T cells from independent donors are shown at the bottom. Statistical significance was determined using a Mann-Whitney U test.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Purification, Incubation, Recombinant, Flow Cytometry, MANN-WHITNEY, Quantitation Assay

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: PBMC were incubated with 2 µg/ml gp120 IIIB (a) or BAL (b) and BSA-Alexa 647 for 2 h at 37°C in the presence of the indicated endocytosis inhibitors (a) or PLA-2 inhibitors (b). Representative flow cytometry histograms showing probe uptake by CD4+ T cells are shown at left. Graphs on the right show the percent probe uptake by CD4+ T cells in the presence of inhibitors compared to the no-inhibitor control from independent donors. Statistical significance was determined using a Student’s 1-sample t-test.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Incubation, Flow Cytometry, Control

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: PBMC were incubated with 2 µg/ml gp120 IIIB (a) or BAL (b,c) or NL4-3 viral supernatants and BSA-Alexa 647 for 2 h at 37°C in the presence of CD4-Ig or sCD4 (a), the indicated anti-Env CD4-binding-site antibodies (b), or the indicated LCK inhibitors (c). Representative flow cytometry histograms of probe uptake in CD4+ T cells are shown at left. Graphs on the right show percent probe uptake in the presence of inhibitors relative to the no-inhibitor control from independent donors. Statistical significance was determined using a Student’s 1-sample t-test.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Incubation, Binding Assay, Flow Cytometry, Control

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: (a) PBMC were incubated with soluble gp120 Envs from HIV-1 clade B (IIIB, BAL, B.MN, B.9021, B.63521, AC1029; blue), clade AE (CM235, 93TH975, AE.244; pink), clade C (CN54, 96ZM651; orange), an HIV-1 main (M) group consensus gp120 (M.CONS; gray), or SIV-1 Env (SIV; gray) (all 2 µg/ml), together with BSA-Alexa 647 for 2 h at 37°C. Shown is fold induction of BSA uptake in CD4+ T cells from independent donors. (b-f) PBMC were preincubated with soluble Envs (2 µg/ml) at 4°C, washed, and stained with the anti-CD4 D1 antibody, RPA-T4, and the anti-CD4 D3 antibody, OKT4. ( b ) A schematic of the effect of Env interaction with CD4 upon anti-CD4 antibody binding is shown at left. A representative flow cytometry histogram of the influence of B.63521 upon RPA-T4 binding to CD4+ T cells is shown in the middle panel. The graph at right shows the percent RPA-T4 binding after treatment with the indicated Env relative to the control without Env from independent donors. (c) Percent RPA-T4 binding plotted against fold induction of probe uptake in CD4+ T cells for the indicated Envs. (d) The graph shows the percent OKT4 binding after treatment with different Envs relative to the control without Env from independent donors. Statistical significance was determined using a Student’s 1-sample t-test (a,b,d). (e) Percent RPA-T4 binding versus OKT4 binding in CD4+ T cells for the indicated Envs. (f) Percent OKT4 binding versus fold induction of probe uptake in CD4+ T cells. Regression lines are shown with coefficients of determination (c,e,f).

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Incubation, Staining, Binding Assay, Flow Cytometry, Control

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: (a) PBMC were incubated with IIIB, BAL, or B.63521 Env (2 µg/ml) in the presence of 902, 447-52D or 268-D IV anti-V3 loop antibodies together with BSA-Alexa 647 for 2 h at 37°C. A representative histogram showing BAL-induced probe uptake by CD4+ T cells in the presence and absence of 268-D IV is shown at left. The graph at right shows the percent Env-induced probe uptake in the presence of antibodies, relative to the absence of antibodies, across independent donors. (b) PBMC were incubated with Envs (2 µg/ml) in the presence or absence of anti-V3 loop antibodies at 4°C, washed, and stained with the anti-CD4 D1 antibody, RPA-T4, and the anti-CD4 D3 antibody, OKT4. The schematic at left shows the potential effect of anti-V3 loop antibodies on Env inhibition of RPA-T4 binding and increased OKT4 binding. The graph at right shows the percent anti-V3 loop antibody recovery of RPA-T4 binding in CD4+ T cells relative to Env in the absence of anti-V3 loop antibody (baseline) and CD4+ T cells without Env (100 percent recovery) from independent donors. Statistical significance was determined using a Student’s 1-sample t-test (a,b). (c) Plot of recovery of RPA-T4 binding versus percent Env-induced probe uptake for the indicated Envs and anti-V3 loop antibodies. ( d ) Plot of percent recovery of RPA-T4 binding versus percent increase of Env-induced OKT4 binding in the presence of anti-V3 loop antibodies relative to their absence.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Incubation, Staining, Inhibition, Binding Assay

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: (a) PBMC were incubated with IIIB, BAL, or B.63521 Env (2 µg/ml) in the presence of E51 or m9 anti-bridging sheet antibodies and BSA-Alexa 647 for 2 h at 37°C. Shown is the percent Env-induced probe uptake in the presence of antibodies, relative to the absence of antibodies, across independent donors. ( b ) PBMC were incubated with Envs (2 µg/ml) in the presence or absence of anti-bridging sheet antibodies at 4°C, washed, and stained with the anti-CD4 D1 antibody, RPA-T4, and the anti-CD4 D3 antibody, OKT4. Shown is the percent anti-bridging sheet antibody recovery of RPA-T4 binding in CD4+ T cells relative to Env in the absence of antibody (baseline) and CD4+ T cells without Env (100 percent recovery) from independent donors. Statistical significance was determined using a Student’s 1-sample t-test (a,b). (c) Plot of recovery of RPA-T4 binding versus percent Env-induced probe uptake for the indicated Envs and anti-bridging sheet antibodies. ( d ) Plot of percent recovery of RPA-T4 binding versus percent increase of Env-induced OKT4 binding in the presence of anti-bridging sheet antibodies relative to their absence.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Incubation, Staining, Binding Assay

Journal: bioRxiv

Article Title: The gp120 Envelope Glycoprotein of HIV-1 Triggers Macropinocytosis in Primary CD4+ T Cells to Promote HIV-1 Infection

doi: 10.64898/2026.04.27.721102

Figure Lengend Snippet: (a) Resting CD4+ T cells from four different donors were inoculated with NL4-3/NanoLuc in the presence of BSA-Alexa 647 for 2 h at 37°C, before washing and determination of probe uptake by flow cytometry. Separate samples of resting CD4+ T cells from the same donors were incubated with NL4-3/NanoLuc without probe for 2 h at 37°C. Cells were then washed and incubated for a further 24 h in the presence of Saquinavir before lysis and assessment of NanoLuc activity by luminescence. Shown are fold increases in probe uptake versus NanoLuc counts for each donor. The regression line with the coefficient of determination is shown. (b) Resting CD4+ T cells were pre-incubated with the fusion inhibitor, T-20, or macropinocytosis inhibitors EIPA, AACOCF3, and FKGK18 for 15 min and incubated with NL4-3/NanoLuc for 2 h at 37°C in the continued presence of macropinocytosis inhibitors and Saquinavir, washed, and cultured for 24 h before lysis and determination of NL4-3/NanoLuc activity. The graph shows NanoLuc activity in the presence of inhibitor relative to the absence of inhibitor from independent donors. Statistical significance was determined using a Student’s 1-sample t-test. (c) Model of mechanism and role of HIV-1-induced macropinocytosis in resting CD4+ T cells. The binding of soluble HIV-1 gp120 Env to the CD4 ectodomain triggers CD4 conformational changes that activate iPLA-2, which converts phospholipids to cone-shaped lysophospholipids on the inner leaflet of the plasma membrane, resulting in inward membrane curvature. Additional signals from CD4 converge upon Rac/Cdc42 and the actin cytoskeleton, which promote further membrane curvature leading to macropinocytosis. Intact HIV-1 virions enter macropinosomes, where HIV-1 fusion with the membrane may be favored, resulting in productive infection.

Article Snippet: Purified resting CD4+ T cells were isolated from PBMC by magnetic negative selection using the Miltenyi Human CD4+ T Cell Isolation Kit (130-096-533) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Lysis, Activity Assay, Cell Culture, Binding Assay, Clinical Proteomics, Membrane, Infection